The vascular endothelium forms a selectively permeable barrier between blood and tissue, and the permeability of this endothelial barrier regulates the transport of cells and molecules between blood and surrounding tissue. Thus, regulation of vascular permeability is fundamental to vascular homeostasis and cardiovascular function, and improper regulation of vascular permeability contributes to the progression of a variety of cardiovascular diseases including stroke, atherosclerosis, inflammation, and myocardial infarction. Hemodynamic shear stress, the frictional force imparted by the flowing blood on the vascular endothelium, is a key regulator of vascular permeability, and disturbed or reduced shear stress results in pathologically increased vascular permeability. Despite the importance of vascular permeability in cardiovascular health and disease, the molecular mechanisms by which shear stress regulates vascular permeability remain poorly understood. In the proposed work, a microvasculature-on-chip model will be implemented to systematically regulate shear stress in physiologically relevant 3D human microvessels and to quantify changes in permeability and vascular structure in response to shear stress and pharmacologic and genetic manipulations. The permeability of the vascular endothelium is principally regulated by intercellular adhesions known as adherens junctions. Although shear stress is known to promote the assembly of adherens junction plaque proteins, the molecular mechanisms by which shear stress regulates the structure and stability of adherens junctions remain unknown. Our preliminary data demonstrate that Notch1, an intercellular receptor critical in vascular development and arterial specification, is upregulated by shear stress and contributes to the stabilization of adherens junctions. Consequently, the proposed work seeks to address the working hypothesis that Notch1 signaling regulates vascular permeability by promoting adherens junction assembly and stability in response to physiologic shear stress through the following specific aims: 1. To investigate whether hemodynamic shear stress regulates vascular permeability through the Notch1 signaling pathway. 2. To investigate the role of Notch1 activation in regulating adherens junction structure and stability. The proposed multidisciplinary approach employs microfluidics, pharmacologic and genetic manipulations, and unbiased proteomic screening to understand the interplay between shear stress, Notch1 signaling, and adherens junction stability. The proposed work will not only elucidate the molecular signaling mechanisms governing vascular permeability, but also establish a new approach for investigating the interactions between the flowing blood and the vascular endothelium.